This invention relates generally to fluorescent dye compounds. More specifically, this invention relates to extended rhodamine dyes useful as fluorescent labeling reagents.
The non-radioactive detection of biological analytes utilizing fluorescent labels is an important technology in modern molecular biology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact and costs associated with reagent disposal is greatly reduced. Examples of methods utilizing such non-radioactive fluorescent detection include automated DNA sequencing, oligonucleotide hybridization methods, detection of polymerase-chain-reaction products, immunoassays, and the like.
In many applications it is advantageous to employ multiple spectrally distinguishable fluorescent labels in order to achieve independent detection of a plurality of spatially overlapping analytes, i.e., multiplex fluorescent detection. Examples of methods utilizing multiplex fluorescent detection include single-tube multiplex DNA probe assays and multi-color automated DNA sequencing. In the case of multiplex DNA probe assays, by employing multiplex fluorescent detection, the number of reaction tubes may be reduced thereby simplifying experimental protocols and facilitating the production of application-specific reagent kits. In the case of multi-color automated DNA sequencing, multiplex fluorescent detection allows for the analysis of multiple nucleotide bases in a single electrophoresis lane thereby increasing throughput over single-color methods and reducing uncertainties associated with inter-lane electrophoretic mobility variations.
Assembling a set of multiple spectrally distinguishable fluorescent labels useful for multiplex fluorescent detection is problematic. Multiplex fluorescent detection imposes at least six severe constraints on the selection of component fluorescent labels, particularly for applications requiring a single excitation light source, an electrophoretic separation, and/or treatment with enzymes, e.g., automated DNA sequencing. First, it is difficult to find a set of structurally similar dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40-80 nanometers (nm). Second, even if dyes with non-overlapping emission spectra are identified, the set may still not be suitable if the respective fluorescent quantum efficiencies are too low. Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are usually widely separated. Fourth, the charge, molecular size, and conformation of the dyes must not adversely affect the electrophoretic mobilities of the analyte. Fifth, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the analyte, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like. Sixth, the dye must have sufficient photostability to withstand laser excitation.
Currently available multiplex dye sets suitable for use in four-color automated DNA sequencing applications require blue or blue-green laser light to adequately excite fluorescence emissions from all of the dyes making up the set, e.g., argon-ion lasers. Use of such lasers in commercial automated DNA sequencing systems is disadvantageous because of their high cost and limited lifetime.
Thus, there exists a need for fluorescent dye compounds which satisfy the above constraints and are excitable by laser light having a wavelength above about 630 nm.
The present invention is directed towards our discovery of a class of extended rhodamine dye compounds suitable for the creation of sets of spectrally-resolvable fluorescent labels useful for multiplex fluorescent detection. The subject dye compounds are particularly well suited for use in automated fluorescence-based DNA sequencing systems using an excitation light source having a wavelength greater than about 630 nm, e.g., a helium-neon gas laser or a solid state diode laser.
In a first aspect, the invention comprises an extended rhodamine compound having the structure 
wherein the composition of moieties R1 through R11, R13, and Y1 through Y4 are as follows. Taken alone, R1 through R7, R9 through R11, and R13 is each independently selected from the group consisting of xe2x80x94H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1, halogen, xe2x80x94OS(O)2OR, xe2x80x94S(O)2OR, xe2x80x94S(O)2R, xe2x80x94S(O)2NR, xe2x80x94S(O)R, xe2x80x94OP(O)O2RRxe2x80x94, P(O)O2RR, xe2x80x94C(O)OR, xe2x80x94NRR, xe2x80x94NRRR, xe2x80x94NC(O)R, xe2x80x94C(O)R, xe2x80x94C(O)NRR, xe2x80x94CN, and xe2x80x94OR. As used here, and throughout this Summary section, each R may be independently xe2x80x94H, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or linking group, and each Z1 may be independently any one of xe2x80x94R, halogen, xe2x80x94OS(O)2OR, xe2x80x94S(O)2OR, xe2x80x94S(O)2R, xe2x80x94S(O)2NR, xe2x80x94S(O)R, xe2x80x94OP(O)O2RRxe2x80x94, P(O)O2RR, xe2x80x94C(O)OR, xe2x80x94NRR, xe2x80x94NRRR, xe2x80x94NC(O)R, xe2x80x94C(O)R, xe2x80x94C(O)NRR, xe2x80x94CN, xe2x80x94O or xe2x80x94OR.
R8 is selected from the group consisting of xe2x80x94H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1.
Taken alone, nitrogen substituents Y1 through Y4 are each independently selected from the group consisting of xe2x80x94H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1.
Alternatively, rather than being taken alone, substituents R1 through R7, R9 through R11, R13, Y1 through Y4 can be taken together in various selected combinations. In particular, R1 may be taken together with R2, Y1 or Y2, R2 may be taken together with R1, R3 may be taken together with R4, R4 may be taken together with R3, Y3 or Y4, R5 may be taken together with R6, Y3 or Y4, R6 may be taken together with R5, R7, Y3 or Y4, R7 may be taken together with R6, R9 may be taken together with R10, R10 may be taken together with R9 or R11, R11 may be taken together with R10, Y1 or Y2, R13 may be taken together with Y1 or Y2, Y1 may be taken together with R1, R11, or Y2, Y2 may be taken together with R1, R11, or Y1, Y3 may be taken together with R4, R5, R6, R13, or Y4, or Y4 may be taken together with R4, R5, R6, R13, or Y3.
In a second aspect, the invention comprises intermediate compounds useful for the synthesis of the extended rhodamine compounds of the first aspect, such intermediates having the structure 
wherein the composition of moieties R1, R2, R9 through R12, Y1 and Y2 are as follows. Taken alone, R1, R2, R9, R10 and R11 is each independently selected from the group consisting of xe2x80x94H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1, halogen, xe2x80x94OS(O)2OR, xe2x80x94S(O)2OR, xe2x80x94S(O)2R, xe2x80x94S(O)2NR, xe2x80x94S(O)R, xe2x80x94OP(O)O2RR, xe2x80x94P(O)O2RR, xe2x80x94C(O)OR, xe2x80x94NRR, xe2x80x94NRRR, xe2x80x94NC(O)R, xe2x80x94C(O)R, xe2x80x94C(O)NRR, xe2x80x94CN, and xe2x80x94OR.
R12 is selected from the group consisting of xe2x80x94H and xe2x80x94C(O)R8, wherein R8 is selected from the group consisting of xe2x80x94H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z1.
Taken alone, nitrogen substituents Y1 and Y2 are each independently selected from the group consisting of xe2x80x94H, alkyl, alkyl independently substituted with one or more Z1, heteroalkyl, heteroalkyl independently substituted with one or more Z1, aryl, aryl independently substituted with one or more Z1, heteroaryl, heteroaryl independently substituted with one or more Z1, arylalkyl, arylalkyl independently substituted with one or more Z1, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z1.
Alternatively, rather than being taken alone, moieties R1, R2, R9, R10, R11, Y1 and Y2 can be taken together in various selected combinations. In particular, R1 may be taken together with R2, Y1 or Y2, R2 may be taken together with R1, R9 may be taken together with R10, R10 may be taken together with R9 or R11, R11 may be taken together with R10, Y1 or Y2, Y1 may be taken together with R1, R11, or Y2, Y2 may be taken together with R1, R11, or Y1.
In a third aspect, the invention comprises nucleotide compounds labeled with the extended rhodamine dyes of the invention, such nucleotides having the structure
xe2x80x83NUCxe2x80x94Lxe2x80x94D
Wherein NUC is a nucleoside/tide or nucleoside/tide analog; L is a linkage; and D is an extended rhodamine dye compound of the first aspect. If NUC comprises a purine base, the linkage is attached to the 8-position of the purine, if NUC comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if NUC comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine.
In a fourth aspect, the invention comprises a fragment analysis method comprising the steps of forming one or more labeled polynucleotide fragments, the fragments being labeled with an extended rhodamine compound of the first aspect; resolving the one or more labeled polynucleotide fragments; and detecting the resolved labeled polynucleotide fragments.
Various aspects and/or embodiments of the above-described invention may achieve one or more of the following important advantages over known fluorescent dye compounds useful for multiplex fluorescent detection: (1) the subject dye compounds may be efficiently excited by a low-cost red laser using wavelengths at or above 630 nm; (2) the emission spectra of the subject dye compounds can be modulated by minor variations in the type and location of nitrogen and/or aryl-substituents, allowing for the creation of dye sets having similar absorption characteristics yet spectrally resolvable fluorescence emission spectra; (3) the subject dye compounds may be easily attached to nucleosides/tides or polynucleotides without compromising their favorable fluorescence properties; (4) the subject dye compounds have narrow emission bandwidths, i.e., the emission bandwidth has a full-width at half the maximum emission intensity of below about 70 nm; (5) the subject dye compounds are highly soluble in buffered aqueous solution while retaining a good quantum yield; (6) the subject dye compounds are relatively photostable; and (7) the subject dye compounds have relatively large extinction coefficients, i.e., greater than about 50,000.
These and other features and advantages of the present invention will become better understood with reference to the following detailed description and appended claims.